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Self-Assembly of Short Peptides

Self-Assembly of Short Peptides. β -Helix Based Membranes. Christian Dittrich, 26/02/07. Introduction/Project. Outline. Use of short peptides for membrane self-assembly Precision (folding) Control of intermolecular interactions (point mutations) Mechanical properties

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Self-Assembly of Short Peptides

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  1. Self-AssemblyofShort Peptides β-Helix Based Membranes Christian Dittrich, 26/02/07

  2. Introduction/Project Outline • Use of short peptides for membrane self-assembly • Precision (folding) • Control of intermolecular interactions (point mutations) • Mechanical properties • Biological functionality • No polymeric character • Secondary structure is key Holowka, E. P., D. J. Pochan, et al. (2005). "Charged Polypeptide Vesicles with Controllable Diameter." Journal of the American Chemical Society 127(35): 12423-12428.

  3. Introduction/Gramicidin Gramicidin • 15mer antibiotic peptide derived from Bacillus brevis • Alternating D- and L-amino acids • β-helical structure (~6 AA per turn!) • Forms cation channels in lipid membranes formyl-L-X-Gly-L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val-L-Trp-D-Leu-L-Y-D-Leu-L-Trp-D-Leu-L-Trp-ethanolamine Glowka, M.L.,  Olczak, A.,  Bojarska, J.,  Szczesio, M.,  Duax, W.L.,  Burkhart, B.M.,  Pangborn, W.A.,  Langs, D.A.,  Wawrzak, Z. Structure of Gramicidin D-Rbcl Complex at Atomic Resolution from Low-Temperature Synchrotron Data: Interactions of Double-Stranded Gramicidin Channel Contents and Cations with Channel Wall Acta Crystallogr.,Sect.D v61 pp.433 , 2005 Lomize, A.L.,  Orekhov, V.I.u.,  Arsen`ev, A.S. Refinement of the spatial structure of the gramicidin A ion channel Biol.Membr.(USSR) v18 pp.182-200 , 1992

  4. Methods/Library Peptides WT formyl-X-G-A-DL-A-DV-V-DV-W-DL-Y-DL-W-DL-W-ethanolamine gA-K2H-K2-L-G-A-DL-A-DV-V-DV-W-DL-W-DL-W-DL-W-NH2 gA-K4H-K-G-K3-L-G-A-DL-A-DV-V-DV-W-DL-W-DL-W-DL-W-NH2 gA-K6H-K3-G-K3-L-G-A-DL-A-DV-V-DV-W-DL-W-DL-W-DL-W-NH2 gA-K8H-K2-G-K3-G-K3-L-G-A-DL-A-DV-V-DV-W-DL-W-DL-W-DL-W-NH2 gA-K10H-K-G-K3-G-K3-G-K3-L-G-A-DL-A-DV-V-DV-W-DL-W-DL-W-DL-W-NH2

  5. Methods/Purification Synthesis / Purification / Analysis • Synthesis on solid phase using Fmoc chemistry • 2 x preparative RP-C18 (TFA, AcOH) • Lyophilization with NH3 (AcOH↑) • Counter-ion free samples gA-K6 gA-K6 Anayltical HPLC, RP-C18 MALDI-TOF-MS

  6. Methods/Self-Assembly Self-Assembly • Ethanol as solvent, Cl- as counterion • Dialysis → H2O (CE, 1 kDa cutoff) • No sizing (sonication, extrusion)

  7. Results/TEM Transition Electron Microscopy (stained) gA-K8

  8. Results/AFM Atomic Force Microscopy • Mica • Tapping mode • Cantilever: 42 N/m • Solid/Air gA-K8

  9. Results/SEM Scanning Electron Microscopy gA-K8

  10. Results/Functionality pH-Dependence (pH 7) 90°; 5 min 2nd order cumulant fit: r = 133.15 nm PdI = 0.196

  11. Results/Functionality pH-Dependence (pH 12)

  12. Results/Functionality pH-Dependence (pH 7) 2nd order cumulant fit: r = 131.78 nm Pdi = 0.174

  13. Structural Conclusions Hydrophobic Effect vs. Aromatic Interactions „Perhaps most importantly, the special complementarity between protein and lipid in the LPPC-gA bilayer leads to a supramolecular organization in which protein rotational diffusion is many orders of magnitude slower than the rotational diffusion of the lipid molecules.“ Macdonald, Seelig Dynamic properties of gramicidin A in phospholipid membranes Biochemistry, 1988 H-L-G-A-DL-A-DV-V-DV-W-DL-W-DL-W-DL-W-NH2

  14. The „Trunk“ H-L-G-A-DL-A-DV-V-DV-W-DL-W-DL-W-DL-W-NH2

  15. More Synthesis The „Trunk“ WT formyl-X-G-A-DL-A-DV-V-DV-W-DL-Y-DL-W-DL-W-ethanolamine gA-K2H-K2-L-G-A-DL-A-DV-V-DV-W-DL-W-DL-W-DL-W-NH2 gA-K4H-K-G-K3-L-G-A-DL-A-DV-V-DV-W-DL-W-DL-W-DL-W-NH2 gA-K6H-K3-G-K3-L-G-A-DL-A-DV-V-DV-W-DL-W-DL-W-DL-W-NH2 gA-K8H-K2-G-K3-G-K3-L-G-A-DL-A-DV-V-DV-W-DL-W-DL-W-DL-W-NH2 gA-K10H-K-G-K3-G-K3-G-K3-L-G-A-DL-A-DV-V-DV-W-DL-W-DL-W-DL-W-NH2 Trunk-K1H-K1-W-DL-W-DL-W-DL-W-NH2 Trunk-K2H-K2-W-DL-W-DL-W-DL-W-NH2 Trunk-K3 H-K3-W-DL-W-DL-W-DL-W-NH2 Trunk-K4H-K4-W-DL-W-DL-W-DL-W-NH2

  16. Results/SEM/Trunk SEM Trunk-K3

  17. Results/CD/Library Variation of b-Lys

  18. Results/CD/Temperature Temperature Dependence gA-K8

  19. Results/CD/Temperature Thermal Stability Trunk-K3 gA-K8 gA-K8 Trunk-K3

  20. Conclusion/Structure Proposed Steps to Membrane Formation Folding Dimerization Self-assembly Random coil Primary structure β-helical 2nd structure Secondary structure Helical dimer 1st order quaternary structure Membrane, Micelles? 2nd order quaternary structure

  21. Conclusions/Overview Conclusions • Efficient synthesis and purification methods of gramicidin derivatives • Membrane formation in water by short peptides (8-28 AA) • Self-contained and robust (hydrophilic block length) formation of monodisperse vesicles by dialysis • Vacuum stable membranes • Functionality by pH-dependent and reversible membrane disintegration (release of vesicle content) • β-helical secondary structure • 1st order quaternary structure: Helical dimerization • 2nd order quaternary structure: Membrane self-assembly by lateral dimer interactions

  22. Macromolecular Colloquium Freiburg • „Induced“ self-assembly • Poly-lysine / amphiphilicity • Purpose • Dissolved in lipid double layers • Apart from biological function? • Rafts? • Adaptability

  23. Acknowledgements • Prof. W. Meier • Dirk de Bruyn • Thomas Schuster • Diana Sebök • Michael Kümin

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